Hematopoietic stem cell transplantation is the most common cell-based immunotherapy in clinical application that has the potential to cure hematological malignancies and other hematopoietic disorders including autoimmune diseases and immunodeficiencies. Currently, bone marrow is the major source of hematopoietic stem cells used in transplantation;however, the immunogenic nature of bone marrow, the risk of graft versus host disease, and the toxicity of the pre-conditioning regimens required limit bone marrow transplantation to the treatment of life-threatening malignancies. Recently, embryonic stem (ES) cells have emerged as a promising source of hematopoietic stem cells for use in transplantation because they are pluripotent, exhibit low immunogenicity, and have a high propensity to proliferate. Our lab has successfully generated hematopoietic progenitor cells (HPC) from ES cells. Preliminary data show that HPCs are poorly immunogenic in vitro and have the full potential to reconstitute the hematopoietic system in immunodeficient mice. However, the immunobiology of HPCs has not been studied. The long-term objective of this application is to establish the ideal immunological conditions under which ES-derived cells can be transplanted in allogeneic recipients without the use of severe immunosuppressive agents. The objectives of this proposal are to study both the innate and adaptive immune responses directed against transplanted ES-derived HPCs. The central hypothesis is that ES-derived HPCs are poorly immunogenic due to their low expression of MHC antigens and are capable of engrafting across MHC barriers without the need for severe immunosuppressive agents. The Specific Aims are: 1. Determine whether the engraftment of ES-derived HPCs is regulated by NK cells. 2. Determine whether ES-derived HPCs stimulate T and B cell alloresponses. ES-derived HPCs will be transplanted into NK-deficient Rag2-/-Gc-/- and NK-replete Rag2-/- mice to investigate the role of NK cells in regulating HPC engraftment. Sensitization of T and B cell alloresponses after HPC transplantation will be evaluated with skin grafts, ELISPOTS, and sera analysis for anti-HPC antibodies. Additionally, 2C mice transgenic for a CD8 TcR specific for the H-2Ld alloantigen will be utilized to investigate HPC susceptibility to alloreactive CTL. When completed, these studies will provide a characterization of the immune response directed against transplanted ES-derived HPC and may provide further insight into the immunobiology of other ES-derived cellular therapies.